CN1867352A - Therapeutic uses of chemokine variants - Google Patents

Abstract

Variants of homodimer-forming chemokines, such as human CCL2, having a single amino acid substitution in the dimerization interface that alters the pattern of hydrogen bonds and acting as an obligate monomer, can antagonize natural chemokines and have anti-inflammatory activity in vivo. These variants can be used as active ingredient in pharmaceutical compositions for the treatment of inflammatory, autoimmune, or infectious diseases.

Description

The therapeutic of chemokine variants is used

Invention field

The present invention relates to chemokine variants more specifically is the therapeutic application of people CCL2 variant.

Background of invention

Chemotactic factor is that a class is little, the short inflammatory protein of secreting type, and mediated leucocytes is to the directional migration of injury.According to the position of the conservative cysteine of this protein family characteristic, chemotactic factor family structurally can be divided into C-C, C-X-C and C-X ₃-C chemotactic factor, they are attached to (people such as Baggiolini M., 1997 on a series of membrane receptor; Fernandez EJ and Lolis, 2002).

These membrane receptors all are seven spiral G protein coupling receptors, and they make chemotactic factor can exercise its biologic activity on target cell, and described target cell can have the particular combinations of these receptors according to its state and type.The physiological action of these chemotactic factors derives from simultaneous interactional complexity and comprehensive system: these receptors have eclipsed ligand specificity usually, thereby a receptor can be in conjunction with different chemotactic factors, and a chemotactic factor also can be in conjunction with different receptors.

Usually chemotactic factor produces in the injury, causes leukocytic migration and activation, has brought into play important function in inflammatory, immunity, stable state, hemopoietic and angiogenic growth process.Promptly use chemotactic factor to have some potential shortcomings (specifically to be as healing potion, coagulation tendency and miscellaneous combination are arranged), but carrying out therapeutic for the disease relevant with said process interferes, these molecules still are considered to good target candidate molecules, by suppressing specificity chemotactic factor and receptor thereof to prevent leukocyte overrecruitment and activation (Baggiolini M, 2001; Loetscher P and Clark-LewisI, 2001; Godessart N and Kunkel SL, 2001).

For the research of structure-activity relationship explanation chemotactic factor two main and sites its acceptor interaction are arranged, pliability amino terminal region and be positioned at the ring of the rigid conformation after second cysteine.It is believed that chemotactic factor passes through this and encircles regional anchor on receptor, and think that this contact helps the combination of amino terminal region, and this combination causes the activation of receptor.Modified by the test amino terminal region or the natural and synthetic chemotactic factor of truncate has proved the importance of amino terminal region.Aminoacid by proteolytic digestion, undergo mutation or chemical modification after, this process activates these molecules or makes these molecular inactivations, produces the chemical compound with excitement and/or antagonistic activity.Therefore, there is the chemotactic factor of specific modification to have the potential (Schwarz and Wells, 1999) of treatment inflammatory and autoimmune disease at amino terminal region.

CCL2, be also referred to as monocyte chemoattractant protein 1 (MCP-1) or MCAF (MCAF), identified in inflammation, to have important function, can promote mononuclear cell and lymphocyte in response to the injury in multiple inflammatory diseases, different types of tumors, heart allograft, AIDS and the pulmonary tuberculosis and infection signal and raise (Yoshimura T etc., 1989; Gu L etc., 1999).Carried out broad research with the various transgenic animal pair physiologically active relevant with CCL2, proved not only monocytic the raising of control in inflammatory model of this chemotactic factor, but also control is reacted relevant cytokine and cytokine expression (Gu L etc., 2000 relevant with arteriosclerotic generation with t helper cell; Gosling J etc., 1999; Lu B etc., 1998).

Say on the structure that CCL2 is made up of with the eclipsed β lamella of α spiral the ring and the C-end of N-end, it forms homodimer, but is detected as monomer (Handel T etc., 1996 under specific experiment condition; KimKS etc., 1996; Lubkowski J, etc., 1997).For a lot of other chemotactic factors, a lot of structures-active case study is provided on the document, wherein by express the N-terminal truncate or in single site alternate variant to take place produced the CCL2 mutant, with estimate lacked or alternate aminoacid in effect (Gong J and Clark-LewisI, 1995 in conjunction with active and other properties relevant with CCL2; Zhang Y etc., 1996; Steitz SA etc., 1998; Gu L etc., 1999; Hemmerich S etc., 1999; Seet BT etc., 2001).

Particularly, studied the effect of dimerization in CCL2 receptors bind and activation, the difference sudden change that the result is presented at the obstruction dimerization that stub area takes place can change some activity of CCL2, as the inhibition of receptor binding affinity, chemotactic stimulation, adenyl cyclase and the stimulation that calcium current is gone into (Paavola C etc., 1998).Yet, a kind of mutant that Paavola describes, this paper is called P8A ^*, dimerization does not take place, but has kept primary effectiveness and efficient, the another one obligate monomer mutant that Paavola describes, this paper is called Y13A ^*, proved its a little less than external binding affinity 100 times, external a little less than being one many ACI and calcium currents are gone into stimulant, and can not be at cell culture moderate stimulation chemotactic.With Y13A ^*Similar, a mutant, [1+9-76] MCP-1 (a kind of CCL2 variant that lacks residue 2-8) is in the activity of external antagonism CCL2.

In an experimental model, based on identification to viral chemokine binding protein M3, also studied contain the alternate CCL2 mutant of P8A in conjunction with feature, the result proves that this virus protein is efficiently in conjunction with this CCL2 mutant (Alexander JM etc., 2002).And having proved the monomer variant such as the CCL2-P8A of chemotactic factor does not have activity in vivo, but its external be activatory fully and have no difference with dimeric forms (Proudfoot A etc., 2003).

Also described the CCL2 mutant example that relates to the residue that does not influence product albumen dimer pattern in the document, it has obtained that CCL2 is had the molecule of antagonism, and (US 5739103, W003/84993).Yet, there is not a kind of so concrete chemotactic factor of hint (specifically being the CCL2 mutant) to can be used as chemokine antagonists in the prior art, this chemotactic factor is because the replacement (as relating to proline) in single site and as a kind of monomer of obligate.

Summary of the invention

Astonishing discovery, a kind of variant of the chemotactic factor (as CCL2) that forms homodimer is the natural chemotactic factor of antagonism and have anti-inflammatory activity in vivo, this variant has an aminoacid replacement that changes the hydrogen bond pattern on dimerization effect interface, be incorporated into receptor and at external obligate monomer with excitability thereby produce.

Variant useful as drug as herein described.Comprise the polypeptide of SEQ ID NO:2 and comprise SEQ ID NO:2 and at 64 polypeptide useful as drug that an isoleucine mutation (SEQ ID NO:4) arranged of SEQ ID NO:2.

Variant as herein described can be used as the medicine for the treatment of autoimmune disease, inflammatory diseases or infectious disease.Comprise the polypeptide of SEQ ID NO:2 and comprise SEQ ID NO:2 and have the polypeptide of an isoleucine mutation (SEQ ID NO:4) to can be used as the medicine of autoimmune disease, inflammatory diseases and infectious disease 64 of SEQ ID NO:2.In one embodiment, comprise the polypeptide of SEQ ID NO:2 and comprise SEQ ID NO:2 and have the polypeptide of an isoleucine mutation (SEQ IDNO:4) to can be used as the medicine for the treatment of multiple sclerosis 64 of SEQ ID NO:2.

Variant as herein described also can be used in the method for treatment autoimmune disease, inflammatory diseases and infectious disease.These methods comprise the monomer variant of the present invention for the treatment of effective dose, and wherein said variant is from least one amino acid replacement, and this substitutes and has changed described chemotactic factor dimerization effect hydrogen bond pattern at the interface.The example that can be used for this monomer variant in these class methods is: the polypeptide that a) comprises SEQ ID NO:2; B) comprise the polypeptide of SEQ ID NO:4; C) a) or b) active mutant; D) comprise a), b) or c) and the polypeptide that belongs to the proteic aminoacid sequence except that described chemotactic factor; And the corresponding molecule of forms such as their active fragment, precursor, salt, derivant, complex or conjugate.

The present invention also comprises pharmaceutical composition, and it contains the polypeptide that comprises obligate monomer antagonism chemokine variants of the present invention.

The present invention also comprises fusion rotein, it contains the obligate monomer antagonism chemokine variants as herein described that is blended in non-chemokine protein sequence, for example the fusion rotein (SEQ ID NO:5) of human immunoglobulin heavy chain's constant region and aminoacid sequence SEQ ID NO:2.The present invention also comprises the method for preparing these fusion rotein.

The present invention also comprises the method for screening obligate monomer antagonism chemokine variants as herein described, comprising: a) produce a single point sudden change of preventing its dimerization ability in CCL2; B) identify the external described variant that is incorporated into receptor and demonstrates excitability; C) from b) the colony that identifies further evaluation can suppress the described variant that peritoneal cell is raised.

From the following detailed description, other characteristics of the present invention and advantage will be very obvious.

Description of drawings

Fig. 1: ripe CCL2 (the SWISSPROT Acc.N of people ^OThe residue 24-99 of P13500; SEQ ID NO:1), ripe CCL2-P8A (SEQ ID NO:2), ripe CCL2 ^*(SEQ ID NO:3), ripe CCL2 ^*The aminoacid sequence of-P8A (SEQ ID NO:4).At CCL2 ^*And CCL2 ^*Add frame table among the-P8A and show that the 64th in aminoacid sports isoleucine (M64I); Corresponding sudden change among the P8A is represented with runic and underscore

Fig. 2: raise in the test cell of recombined human CCL2 and CCL2-P8A raised at peritoneal cell and actively compare with baseline values

Fig. 3: the expression peritoneal cell is raised CCL2-P8A dose response inhibition activity in the test, in this test with two kinds of different inducers: CCL2 (A) or thioglycolate salt (B); With these results and baseline values, negative contrast (having only carrier, saline) with over against according to (dexamethasone, Dex; Only in B) relatively.

Fig. 4: the inhibition activity of CCL2-P8A in the inductive lung inflammation test of expression ovalbumin; The result is compared with baseline and negative contrast (saline).

Fig. 5: be illustrated in the multiple sclerosis animal model of a kind of EAE of being called (experimental autoimmune encephalomyelitis), when the disease degree of animal is that (low clinical scores is not more than 2 to moderate; A) or seriously (high clinical scores is 3 at least; B) time, the effect of CCL2-P8A in reducing clinical scores; With data with only with carrier (negative contrast) or over against according to (the mouse interferon β that is used for the EAE model) time observed data compare; The significance,statistical level of observed effect (was calculated with one-way ANOVA when the numerical statement of each figure bottom asterisk was shown in each time point with negative comparing, carry out the Fisher test then, * represent p＜0.05, * * represents p＜0.01, and * * * represents p＜0.001).

Fig. 6: when giving CCL2-P8A with various dose in the expression animal model, CCL2-P8A is to the inhibition activity of scytitis (the inductive inflammation on the sensitized mice ear of DNFB); CCL2-P8A handles the influence of back to ear swelling volume, and compare: not sensitization but the animal (A) attacked with DNFB at the 5th day with following contrast, sensitization and attack with DNFB but only use the animal (B) of vehicle treated and the animal (Dex that handles with dexamethasone; With with the administration of CCL2-P8A the same manner).(natural law after handling and attacking) detected numerical value in the 6th day.

Fig. 7: by single/a plurality of aminoacid addition and/or substitute (A) or merge (B by residue 243-474 with human normal immunoglobulin λ heavy chain IgG1; What add double underline is the CCL2 signal sequence, and underlined is ripe CCL2-P8A) and the CCL2-P8A of other form of producing.

Detailed Description Of The Invention

By the evidence of above-mentioned prior art as can be known, do not point out a kind of CCL2 variant in the prior art, this variant is produced by dimerization effect at least one amino acid replacement at the interface, thereby should substitute change hydrogen bond pattern and produce the external obligate monomer that is incorporated into acceptor and has excitability, do not point out in vivo antagonism CCL2 of a kind of like this CCL2 variant in the prior art, and capable of inhibiting cell raising and/or inflammatory reaction generally speaking.

In the ripe CCL2 of people, a described amino acid replacement is preferably the 8th of amino acid residue, specifically 8 replaces proline by alanine in the position. These variants do not contain other sudden change on the 9th, 10 or 13 the position of the ripe CCL2 corresponding to the people.

An above-mentioned alternative example is the monomer variant that is called the CCL2 mature form of CCL2-P8A, it can be expressed as the single replacement that proline is replaced with alanine (SEQ ID NO:2) at the 8th, or comprise that again another one replaces (promoting this protein expression), be about to methionine 64 and replace with isoleucine (SEQ ID NO:4). The 8th upper proline replaces with alanine (this amino acid has different chemical group directions, may form hydrogen bond) and produced the CCL2 variant as the obligate monomer. CCL2-P8A and CCL2^*-P8A can think to have active molecule of equal value.

Specificity obligate monomer variant for these CCL2 (is called CCL2-P8A and CCL2^*-the pharmaceutical applications, the method and composition that P8A) it is contemplated that can expect that also activated form under CCL2 or other nature is that other obligate monomer variant that produces with the same manner of dimeric chemotactic factor (CF) also can be used in these pharmaceutical applications, the method and composition. These mutant should have antagonism and the rejection with CCL2-P8A and the natural chemotactic factor (CF) of the similar antagonism of CCL2*-P8A, thereby can medically use.

One aspect of the present invention is the application of the obligate monomer variant of the chemotactic factor (CF) that forms homodimer (CCL2 for example, known it be the therapeutic target of various diseases such as autoimmune disease, inflammatory disease or infectious diseases). These variants derive from described chemotactic factor (CF) dimerization effect at least one 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor at the interface, this replacement has changed the hydrogen bond pattern, thereby produced and externally be incorporated into acceptor and have excitability and also have in vivo the obligate monomer of Antagonism, it as active component, specifically is used for the treatment of or preventing autoimmune disease, inflammatory disease or infectious diseases in pharmaceutical composition. In the test that allows assess performance (as the effect of in peritonaeum, raising cell after induced by inflammatory molecule (chemotactic factor (CF) such as CCL2 itself, thioglycolate salt or ovalbumin)), can obviously see Antagonism in these bodies.

These obligate monomer variants can derive from dimerization effect at least one 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor at the interface, and this replacement has changed the hydrogen bond pattern. More preferably, because proline is the known amino acid relevant especially with setting up three-dimensional single-minded hydrogen bond (relevant with protein complexes such as the dimeric formation of homotype), so the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor proline of the non-proline of described monamino acid Replacing using. Perhaps, described monamino acid Replacing using proline replaces the amino acid of non-proline, thereby changes the stereocpecificity of hydrogen bond.

Other form of the monomer variant of the chemotactic factor (CF) of the above-mentioned formation homodimer that can be used as active component in pharmaceutical composition comprises:

E) active mutant; Or

B) comprise the polypeptide of following material: described variant or described active mutant, and belong to described

Change the aminoacid sequence of the different protein sequence of the factor.

Term " activity " refers to that this optional chemical compound should keep the functional characteristic of CCL2 mutant of the present invention, i.e. antagonism CCL2 and suppress that cell is raised and/or inflammatory reaction in vivo.

Obligate monomer chemokine antagonists as herein described (as reactive compound of the present invention) can be forms such as its active fragment, precursor, salt, derivant, complex or conjugate.

These optional chemical compounds are included in the molecule that the variation of fundamental characteristics disclosed herein has taken place not influence in the sequence of monomer variant of the chemotactic factor that forms homodimer.For example, at a kind of mutant (CCL2-M641 or the CCL2 of CCL2 ^*, kept normal CCL2 active but aspect recombinant expressed performance be improved) the basis on produced CCL2 ^*-P8A.

The antagonism (CCL2-P8A that is used as the CCL2 antagonist herein is as illustration) of monomer variant that forms the chemotactic factor of homodimer as mentioned above remains in active mutant even is strengthened.This quasi-molecule comprises the natural or synthetic analog of described sequence, wherein one or more aminoacid sequences are added, lack or replace, prerequisite is when measuring with disclosed method among the known in the art and following embodiment, they have same biologic activity described herein, and it is on close level or is higher.

Natural analog is meant the human chemokine albumen that identifies in other organism, as mice CCL2 (SWISSPROT Acc.N ^OP10148), corresponding sequence.The artificial analog of monomer variant that forms the chemotactic factor of homodimer is meant with known chemosynthesis and/or site-directed mutagenesis technique, the polypeptide studying or prepare with other suitable known technology in the combination technique (as DNA reorganization, phage display/selections) of dna encoding sequence level, with computer-aided design, these technology provide the sudden change of basic correspondence of finite aggregate or the peptide or the polypeptide of truncate, and persons skilled in the art can obtain and check these peptides and polypeptide routinely with the technology that provides among prior art and the following embodiment.

For example, specific artificial mutant can have one or more aminoacid to be substituted and described one or more aminoacid and generation CCL2 antagonist relevant (WO03/84993) in other position of CCL2.

According to the present invention, the preferred variation is commonly referred to as " guarding " or " safety " replacement in active mutant, relates to non-basic aminoacid.Aminoacid during conservative amino acid replaces has enough similarly chemical property, with structure and the biological function that keeps this molecule.Be clear that very much, also can in above-mentioned sequence, carry out aminoacid insertion and disappearance and its function that do not change, if especially insert and lack and only relate to a few amino acids, for example, be less than 10, preferably be less than 3, and do not remove or replace the vital aminoacid of functional conformation for albumen or polypeptide.

A lot of models are provided in the document, can be based on selecting conservative amino acid to replace (Rogov SI and NekrasovAN, 2001) on these models to the sequence of native protein and/or statistics and physics and chemistry research of structure.The protein contrived experiment proves, adopt the aminoacid of specific subgroup can produce collapsible and activated protein, the classification that helps aminoacid " synonym " to replace, described " synonym " replacement is easier to be held by protein structure, and can be used for detection functionality and structural congener and likeness in form thing (Murphy LR etc., 2000).Simultaneous administration and preferred synonym group are those that define in the Table I.For example, used similar standard at CCL2 ^*And CCL2 ^*Selected methionine among the-P8A with 64 of isoleucine replacements.

Another group active mutant of the chemotactic factor monomer variant of above-mentioned formation homodimer is that polypeptide is intended like thing (be also referred to as and intend peptide), and wherein the essence of peptide or polypeptide has chemical modification in the main chain level of amino acid side chain, amino acid chiral and/or peptide.The purpose of these changes provides the chemotactic factor monomer variant that has the formation homodimer of similar or improvement characteristic at aspects such as preparation, effect and/or pharmacokinetics features.

For example, there is a problem to be exactly, after peptide is being injected into object,, intends replacing responsive especially peptide bond with the peptide that can not rupture and just can make peptide more stable so more useful as therapeutic agent like thing easily by the peptide enzymatic breaking.Similarly, replacing the L-amino acid residue is to make peptide more insensitive and finally more be similar to organic compound but not a kind of standard method of polypeptide to Proteolytic enzyme.Other usefully amino terminal prevent group; as tert-butoxycarbonyl, acetyl group, theyl, succinyl group, methoxyl group succinyl group, suberyl, adipyl, nonanedioyl (azelayl), dansyl base, benzyloxycarbonyl group, fluorenyl methoxy carbonyl, methoxyl group nonanedioyl, methoxyl group adipyl, methoxyl group suberyl and 2,4-dinitrophenyl.Also known a lot of other the made effects in this area increase, actively prolong, purification and/or increase the modification (WO02/10195 of half-life easily; Villain M etc., 2001).

To intend like amino acid whose preferred other " synonym " group in the thing be shown in the Table II those for being included in peptide.Some non exhaustive examples of amino acid derivativges also comprise aminoisobutyric acid (Aib), hydroxyproline (Hyp), 1,2,3,4-tetrahydrochysene-isoquinolin-3-COOH, indoline-2 carboxylic acid, 4-two fluoro-proline, L-Thiazolidine-4-carboxylic acid, the high proline of L-, 3,4-dehydrogenation-proline, 3,4-dihydroxy-phenylalanine, cyclohexyl-glycine and phenylglycine.

" amino acid derivativges " is meant aminoacid or the aminoacid sample chemical entities except that the naturally occurring aminoacid of 20 kinds of genetic codings.Particularly, amino acid derivativges can comprise and replace or unsubstituted linear, branch or annular moieties, and can comprise one or more hetero atoms.Amino acid derivativges can be (Calbiochem-Novabiochem AG, the Switzerland that from the beginning prepares or obtain from commercial channels; Bachem, USA).

Being used for technology synthetic and that the research peptide is intended intending like thing like thing and non-peptide is (HrubyVJ and Balse PM, 2000 well known in the art; Golebiowski A etc., 2001).Also disclose in the document and adopted in the external or body translation system that alpha-non-natural amino acid is mixed albumen to explore and/or to improve the whole bag of tricks (Dougherty DA, 2000) of protein structure and function.

The invention discloses and to form the chemotactic factor monomer variant of homodimer and active mutant thereof as the active component in the pharmaceutical composition, and the albumen that will contain their aminoacid sequence and belong to the aminoacid sequence of the protein sequence except that described chemotactic factor is used as the activity in the pharmaceutical composition.A kind of heterologous sequence in back should provide other characteristic and not hinder its application in pharmacy.Described other characteristic is easier purification, the half-life in body fluid is longer or locate in the extracellular.A kind of feature in described back is extremely important for the specificity grouping of fusion that comprises in the above-mentioned definition or chimeric protein, because it not only is positioned at these monomer variants to help it is separated place with purification, also be positioned at the place of CCL2 and other interaction of molecules under the native state.

Method and strategy (Nilsson J etc., 1997 of structure, purification, detection and the application of the design of part, aglucon, joint and these fusion rotein have extensively been described in the document; " the application Methods Enzymol. 326-328 volume of chimeric protein and hybrid protein, Academic Press, 2000; WO 01/77137).Other protein sequence can be used for producing other form of the chemotactic factor obligate monomer variant of above-mentioned formation homodimer, described other protein sequence is the extracellular domain of embrane-associated protein, the constant region of immunoglobulin (Fc district), multimerization domain, extracellular protein, contain signal peptide albumen, contain the albumen of output signal.The selection that is blended in one or more above-mentioned sequences of described monomer variant is practical for the concrete purposes of described sequence.When described other protein sequence (as the extracellular sequence, contain the albumen of output signal or signal peptide) when making the monomer variant be secreted into extracellular space, because carry out subsequent treatment, this material can more easily be collected and purification from cultured cells, perhaps, can directly use or give these cells.

The obligate monomer variant of the chemotactic factor of above-mentioned formation homodimer also can other preferred form use, for example, and as active fragment, precursor, salt, derivant, conjugate or complex.

Term " fragment " refers to any fragment of chemical compound polypeptide chain itself, independent or bonded with it correlation molecule or the combination of residue (as saccharide residue or phosphate) or the aggregation of original polypeptide or peptide.These molecules also can be modified from other; these are modified and are not generally changed elementary sequence; for example in the body of peptide or external chemical derivatization (acetylation or carboxylation), phosphorylation (introducing phosphorylated tyrosine, phosphorylation serine or phosphorylation threonine residues) by changing peptide in the synthetic and course of processing of peptide or the further processing steps or glycosylation (make peptide with influence glycosylated enzyme for example mammal glycosylase or deglycosylating enzyme contact) pattern.

" precursor " is the chemical compound that gives before or after cell or the health to be converted into by metabolism or enzyme process The compounds of this invention.

The term of this paper " salt " refers to the carboxylic salts and the amino acid-addition salts of peptide of the present invention, polypeptide or its analog.Available methods known in the art prepare carboxylic salts, and carboxylic salts comprises inorganic salt, for example sodium, calcium, ammonium, ferrum or zinc salt etc., and the salt that forms with organic base, for example, the salt that forms with amine such as triethanolamine, arginine or lysine, piperidines, procaine etc.Acid-addition salts comprises, for example, and the salt that forms with mineral acid example hydrochloric acid or sulphuric acid, and the salt that forms with organic acid such as acetic acid or oxalic acid.Any of these salt all should have similar substantially activity with polypeptide or their analog to peptide of the present invention.

Term used herein " derivant " refers to known method from the side chain of amino acid moiety or the functional groups that exists of N-or C-end group and the derivant for preparing.These derivants comprise that for example the ester of carboxyl or aliphatic amide are swum `` from the N-of amino acyl derivative, or the O-acyl derivative of free carboxy, and they and acyl group (for example alkanoyl (alcanoyl) or aroyl) form.

Available molecule known in the art and method, with receptor or other albumen (radioactivity or fluorescent labeling, biotin), molecule (as Polyethylene Glycol and other natural or synthetic polymer) (Harris JM and Chess RB, 2003 that have therapeutic efficiency (cellulotoxic preparation) or improve the drug conveying efficient of described monomer variant; Greenwald RB etc., 2003; Pillai O and Panchagnula R, 2001) produce the useful conjugate or the complex of obligate monomer variant of the chemotactic factor of above-mentioned formation homodimer.Residue with flexible side-chains of can be used for polymer and connecting (that is the side chain that, has the aminoacid of functional group such as lysine, aspartic acid, glutamic acid, cysteine, histidine etc.) can be used for connecting.The residue that perhaps, can replace these positions with a different aminoacid with the flexible side-chains that can be used for the polymer connection.The amino acid whose side chain chemical modification of genetic coding can be connected to be used for polymer, or use alpha-non-natural amino acid with suitable functional side chain group.The polymer connection not only can be connected on the naturally occurring amino acid side chain of antagonist particular location, or be connected on the side chain of the naturally occurring amino acid whose natural or alpha-non-natural amino acid that replaces the antagonist particular location, also can be connected in sugar or other parts (they are connected with the amino acid side chain of target position).

The polymer that is applicable to these purposes is a biocompatibility, that is, they are avirulent for biosystem, known a lot of this polymer.These polymer can be hydrophobic or hydrophilic, biodegradable, not biodegradable or these combination.These polymer comprise natural polymer (as collagen, gelatin, cellulose, hyaluronic acid) and synthetic polymer (polyester, poe, polyanhydride).The example of the nondegradable polymer of hydrophobicity comprises polydimethylsiloxane, polyurethane, politef, polyethylene, polrvinyl chloride and polymethyl methacrylate.The example of the nondegradable polymer of hydrophilic comprises poly-(2-methylol ethyl acrylate), polyvinyl alcohol, poly-(N-vinylpyrrolidone), poly-alkane (polyalkylenes), polyacrylamide and their copolymer.Preferred polymer comprises the sequence repetition unit ethylene oxide,1,2-epoxyethane, as Polyethylene Glycol (PEG).

The synthetic combination that is connected with chemistry of peptide is adopted in preferred method of attachment.It is favourable that water-soluble polymer is connected (especially connecting at proteic amino terminal region) by biodegradable joint.This modification can provide the albumen of precursor forms (prodrug), can discharge albumen when joint is degraded and need not modify polymer.

As conventional method, the obligate monomer variant of the chemotactic factor of above-mentioned formation homodimer can prepare with any method known in the art, comprises recombinant DNA correlation technique and chemical synthesising technology.

A lot of books and summation have been instructed and how to have been used carrier and protokaryon (as escherichia coli) or eukaryotic host cell to clone and prepare recombiant protein, as some chapters and sections (" dna clone 2: expression system ", 1995 in " practical approach " (" APractical Approach ") series of Oxford University Press publication; " dna clone 4: mammlian system ", 1996; " protein expression ", 1996; " purified technology of protein ", 2001).

Another embodiment of the present invention is the nucleotide sequence of coding obligate monomer chemokine variants antagonist as herein described.

The DNA sequence of the obligate monomer variant of the chemotactic factor of coding formation homodimer can be inserted and be connected in suitable free or non-homogeneous/homologous integration vector, it can (transform by any suitable manner, transfection, coupling, protoplast merges, electroporation, calcium phosphate precipitation, direct microinjection etc.) transform and be incorporated in the appropriate host cell.For selecting specific plasmid or the important factor of viral vector to comprise: the easy degree of from carrier-free those recipient cells, discerning and select the recipient cell that comprises carrier; The copy number of desirable carrier in specific host; And whether wish that carrier " shuttles back and forth " between two kinds of dissimilar host cells.

The fusion rotein that described carrier should allow isolating albumen or comprise antagonist of the present invention is regulated in transcription initiation/termination under the control of sequence and is expressed in prokaryotic cell or eukaryotic cell, and described carrier is selected as in described cell constitutive activity or derivable.The cell line of described cell of having separated abundant enrichment then, thus a kind of stable cell line is provided.

For eucaryon host (as yeast, insecticide or mammalian cell), can use the different adjusting sequences of transcribing and translate, this depends on host's character.They can be viral sources, and as adenovirus, bovine papilloma virus, simian virus or similar, wherein conditioning signal is relevant with the specific gene with high expression level.The TK promoter that herpesvirus is for example arranged, the early promoter of SV40, yeast ga14 gene promoter etc.The transcription initiation conditioning signal may be selected to and allows to check and activate, and is convenient to the expression of regulator gene.The cell of the DNA of stable conversion introducing can be selected by introducing one or more labellings, and described labelling allows to select the cell that comprises this expression vector.Described labelling also can be for the auxotroph host provides phototrophy, biocide resistance, as antibiotic or heavy metal such as copper etc.Selectable marker gene can be directly connected on the DNA gene order that needs to express, or is incorporated in the identical cell by cotransfection.For synthetic proteins best, other element also may need.

Host cell can be protokaryon or eucaryon.Eucaryon host preferably such as mammal such as people, monkey, mouse cell and Chinese hamster ovary (CHO) cell, because they provide post translational modification for protein molecular, comprises correct folding or in correct site glycosylation.Equally, the peptide that yeast cells can be finished after the translation is modified, and comprises glycosylation.Existing many recombinant DNA strategies, they utilize strong promoter sequence and high copy number plasmid, can be used to produce in yeast required albumen.Yeast identifies targeting sequencing in clone's mammalian genes product, and secretes the peptide (that is propetide) that to carry targeting sequencing.

For long-term, high yield recombinant polypeptide product, preferred stable expression.For example, but can be transformed into the expression vector that comprises virus replication starting point and/or endogenous Expression element in the cell line of the required polypeptide of stably express, and on same or the carrier that separates, comprise selectable marker gene.After carrier is introduced, allow cell in nutritious culture medium, grow 1-2 days earlier, transfer on the selective medium again.But the purpose of using selected marker is to carry out resistance to select, but there is the cell growth of feasible successful expression calling sequence in it and is recovered.The resistance clone of the cell of stable conversion can use the tissue culture technique that is suitable for described cell type to breed.Separate the cell line that is rich in described cell then, obtain stable cell line.

The mammal cell line that is used to express as the host is known in the art, comprise many immortalized cell lines that can obtain from American type culture collection (ATCC), include but not limited to: Chinese hamster ovary (CHO) cell, Hela, baby hamster kidney cell (BHK), monkey-kidney cells (COS), C127,3T3, BHK, HEK293, Bows melanoma and human hepatocellular carcinoma (for example Hep G2) cell and a lot of other cell line.In rhabdovirus system, baculovirus/insect cell expression system can kit form commerce be buied, especially " MaxBac " test kit (Invitrogen).

The example of chemical synthesising technology is that solid phase synthesis and liquid phase are synthetic.Solid phase synthesis for example is; the corresponding aminoacid of the C-end of required synthetic peptide connected be attached in organic solvent on the insoluble holder; by alternative reaction repeated; to have by the amino group of suitable blocking group protection and the aminoacid of functional side chain group; according to order from the C-end to the condensation one by one of N-end; the blocking group of the amino group of the aminoacid that is attached to resin or peptide is discharged, and peptide chain extends by this way.Solid phase synthesis process can mainly be divided into tBoc method and Fmoc method, depends on the type of employed blocking group.Normally used blocking group comprises: be used for the tBoc (t-butoxy carbonyl) of amino group, Cl-Z-(2-chlorine benzyloxycarbonyl), Br-Z (2-bromine Bian oxygen base carbonyl), Bzl (benzyl), Fmoc (9-fluorenyl methoxy carbonyl), Mbh (4,4 '-dimethoxy benzhydryl), Mtr (4-methoxyl group-2,3,6-trimethylbenzene sulfonyl), Trt (trityl), Tos (tosyl), Z (benzyloxycarbonyl) and Cl2-Bzl (2, the 6-dichloro benzyl); The NO2 (nitro) and the Pmc (2,2,5,7,8-pentamethyl color alkane-6-sulphonyl) that are used for guanidino group; And the tBu (t-butyl) that is used for oh group.Behind synthetic required peptide, carry out protective reaction and downcut from solid support.In the Boc method, polypeptide excision reaction can be undertaken by fluohydric acid gas or trifluoromethane sulfonic acid, in the Fmoc method, uses TFA.Complete synthesis CCL2 albumen is disclosed in the literature (Brown A etc., 1996).

The purification of the monomer variant of the chemotactic factor of the formation homodimer of synthetic or reorganization mentioned above can realize that promptly the method for any routine comprises: extracting, precipitation, chromatography, electrophoresis, or similar method by any method that is used for this purpose.Can preferentially be used for purification proteic other purification process of the present invention is the affinity chromatograph that adopts monoclonal antibody or affinity groups, and described antibody or affinity groups can be attached to target protein and generate and be fixed on the gel-type vehicle that fills in the post.Comprise described proteic impure prepared product by described post.Described albumen will be attached on the post by heparin or by specific antibody, and impurity will pass the pillar outflow.After washing, by changing pH or ionic strength with described albumen eluting from the gel.Perhaps, can use HPLC (high performance liquid chromatography).Can use the solvent that is usually used in protein purification to come eluting based on water-acetonitrile.

The monomer variant that forms the chemotactic factor of homodimer can be used in the pharmaceutical composition, is used for the treatment of or prevents autoimmune disease, inflammation or catch.Especially, in an embodiment, the result that multiple sclerosis animal model obtains shows that these monomer variants can be used on treatment or the prevention that is used for multiple sclerosis in the pharmaceutical composition.

Another aspect of the present invention is the application that the monomer variant of the chemotactic factor of formation homodimer is used as medicine, wherein said variant is produced by at least one aminoacid replacement, changes the pattern of hydrogen bond on the described dimerization interface that is substituted in described chemotactic factor.The example of described variant is CCL2-P8A and CCL2 in this article ^*-P8A.Yet instruction of the present invention allows to differentiate, produce and test similar molecule based on the sequence of other chemotactic factor with activity.

Particularly, these monomer variants can be selected from:

a)CCL2-P8A(SEQ ID NO：2)；

b)CCL2 ^*-P8A(SEQ ID NO：4)；

C) a) or b) active mutant; Or

D) comprise a), b) or c) and the polypeptide that belongs to the aminoacid sequence of the protein sequence except that described chemotactic factor;

And the corresponding monomer variant that exists with their active fragment, precursor, salt, derivant, complex or conjugate form.

Others of the present invention also comprise a kind of pharmaceutical composition, its monomer variant that comprises a kind of chemotactic factor that forms homodimer is as active ingredient, wherein, described variant is produced by at least one aminoacid replacement, change the pattern of hydrogen bond on the described dimerization interface that is substituted in described chemotactic factor, as CCL2-P8A or CCL2 ^*-P8A, randomly with foregoing any form (, comprise their polypeptide, or conjugate) as active mutant, and encode they DNA and express their cell.

Pharmaceutical composition of the present invention can comprise suitable pharmaceutically acceptable carrier, the carrier of biocompatibility and the additive (as normal saline) that is suitable for delivering medicine to animal, and comprise adjuvant at last (as excipient, stabilizing agent or diluent) so that with reactive compound be processed into can be medicinal preparation.Described pharmaceutical composition can any acceptable manner be prepared, to satisfy the needs of administration.Such as, applying biological material and other polymer are used for medicine to be sent, and uses effectiveness open (Luo B and Prestwich GD, 2001 in the literature that different technology and model are verified the specific administration mode; Cleland JL etc., 2001).

" effective dose " thus be meant and be enough to influence the process of disease and the amount of the active ingredient that alleviates or remove that seriousness causes described disease.Described effective dose depends on route of administration and status of patient.

" pharmaceutically acceptable " is meant and comprises any carrier, the bioactive effectiveness that it can the interferon activity composition, and do not have toxicity for the host of administration.For example, for parenteral, above-mentioned active ingredient can be prepared into unit dosage form, is used for injecting at carrier such as saline, glucose solution, serum albumin and Ringer ' s solution.Carrier can be selected from starch, cellulose, Pulvis Talci, glucose, lactose, sucrose, gel, Fructus Hordei Germinatus, rice, flour, Chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, drying defatted breast, glycerol, propylene glycol, water, ethanol and various oil, comprises it being the oil (Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Oleum sesami) in oil, animal oil, vegetable oil or synthetic source.

Any acceptable form of medication can be used, and determines active ingredient required level in blood by those skilled in the art.For example, can pass through different parenteral administrations, as subcutaneous, intravenous, intradermal, muscle, intraperitoneal, intranasal, percutaneous, oral or buccal approach.Parenteral can pass through bolus infusion, by pouring into gradually or the controlled release dosage form along with the time, comprise store injection (depotiniection), osmotic pumps and analog, in order under predetermined speed, to prolong the administration time of polypeptide, preferably to be suitable for the form of the unit dose that the exact dose single gives.The preparation of parenteral comprises aquesterilisa or non-aqueous solution, suspension and Emulsion, and it can comprise adjuvant known in the art or excipient, and can prepare according to conventional methods.In addition, reactive compound can be suspended in administration in the suitable oil-containing injectable suspensions.The lipophilic solvent or the carrier that are fit to comprise fatty oil such as Oleum sesami, or Acrawax such as Oleum sesami, or synthetic fatty acid ester such as ethyl oleate or triglyceride.Water injection suspension liquid can comprise the material that increases suspension viscosity, for example sodium carboxymethyl cellulose, sorbitol and/or glucosan.Can be randomly, described suspension can comprise stabilizing agent.Pharmaceutical composition comprises the suitable solution that is used for drug administration by injection, and comprises from about 0.01% to 99% preferred 20% to 75% reactive compound and excipient.Described compositions can be comprised suppository by rectally.

The dosage that should be understood that administration depends on receiver's age, health degree and weight, the treatment type (if there is) of carrying out simultaneously, treatment frequency, and the character of the required effect that reaches.Dosage is formulated according to individual subjects, and this is that those skilled in the art can understand and determine.The required accumulated dose of each treatment can give by multi-agent or single agent.Pharmaceutical composition of the present invention can be given separately or be combined with other treatment according to other symptom of the conditioned disjunction state of an illness.Usually, the daily dose of active ingredient is the every kg body weight of 0.01-100 milligram.Usually, gradation every day gives or gives the 1-40 milligram every kg body weight with sustained release form, can reach required effect.For the second time or the dosage of follow-up administration identical, be lower than or be higher than and give individual dosage first or formerly.

The method that another object of the present invention provides a kind of treatment or prevents autoimmune disease, inflammation (as multiple sclerosis) or catch, the monomer variant that comprises the chemotactic factor of the formation homodimer that gives effective dose, wherein, described variant is formed by at least one aminoacid replacement, and this replacement has changed the form of the hydrogen bond on the dimerization interface of described chemotactic factor.The example that can be used to the monomer variant of described method is:

a)CCL2-P8A(SEQ ID NO：2)；

B) CCL2 ^*-P8A mutain (SEQ ID NO:4);

C) a) or b) active mutant; Or

D) comprise a), b) or c) and belong to the polypeptide of the aminoacid sequence of the protein sequence except described chemotactic factor;

And the corresponding monomer variant that exists with their active fragment, precursor, salt, derivant, complex or conjugate form.

A kind of addressed before infinite relate to application of the present invention, the autoimmune disease of variant and method, inflammation or the example that catches are as follows: arthritis, rheumatic arthritis (RA), psoriatic arthritis, osteoarthritis, systemic lupus erythematosus (sle) (SLE), systemic sclerosis, scleroderma, polymyositis, glomerulonephritis, cystic fibrosis, fibroid degeneration, irritated or super quick disease, dermatitis, asthma, chronic pulmonary is blocked disease (COPD), enteritis (IBD), Crohn ' s disease, ulcerative colitis, multiple sclerosis, cancer, septic shock, virus or HIV infect, transplant, airway inflammation (airways inflammation), anti-host disease of transplantability (GVHD) and arteriosclerosis.

The treatment of polypeptide of the present invention and related reagent use can by in the body or in vitro tests use zooblast, tissue and model to evaluate (in the mode of safety, pharmacokinetics and effectiveness) (Coleman RA etc., 2001; Li AP, 2001; Methods Mol.Biol vol.138, " Chemokines Protocols " is by editors such as Proudfoot A, Humana PressInc., 2000; Methods Enzymol, vol.287 and 288, Academic Press, 1997).

Another aspect of the present invention is the method for screening obligate monomer antagonist chemokine variants as herein described, comprising:

A) simple point mutation of its dimerization ability of formation blocking-up in CCL2;

B) be attached to receptor and present the variant of antagonistic properties in external discriminating;

C) from front b) differentiate and to identify further in the group obtain that it suppresses peritoneal cell and raises characteristic.

The assessment of these characteristics can be undertaken by technology known in the art, as shown in embodiment, uses known molecule of inducing inflammation and peritoneal cell to raise, for example a kind of chemotactic factor such as CCL2 self, thioglycolate salt, or ovalbumin.

The present invention describes by the reference specific implementations, need not the implication and the purpose of claim are made to expand the change and the replacement mode that can realize but these descriptions can comprise all those skilled in the art.

The present invention will describe by following embodiment, and these embodiment are not construed as limiting the invention.

Embodiment

Embodiment 1: the clone of recombiant protein, express, and purification

Based on the sequence of the mature form of people CCL2/MCP-1, corresponding to the fragment 24-99 of precursor molecule, as document (Paavola CD etc., 1998) described in, generate mature C CL2 (Fig. 1, SEQ ID NO:1) and CCL2-P8A mutain (Fig. 1, SEQ ID NO:2), mature C CL2 ^*(Fig. 1, SEQ ID NO:3) and CCL2 ^*-P8A mutain (Fig. 1, SEQ ID NO:4), express recombinant protein in E.coli.For CCL2 ^*And CCL2 ^*-P8A replaces methionine with isoleucine on the 64th, by get rid of the formation of the type that comprises methionine-sulfoxide on the 64th, improved the purity and the homogeneity of mutant.

In brief, be used for CCL2 ^*The gene synthesis technology of CCL2 gene by standard make up, express in E.coli with best codon usage, the codon of the methionine of encoding is added on and is encoded into acquaintance CCL2 or CCL2 ^*5 ' end of sequence.The 8th has the mutation construction thing of alanine to pass through with CCL2 or CCL2 ^*Be the polymerase chain reaction formation of template, and be cloned between the Xho I and Nde I site of pET3a plasmid (Novagen).

Use coding CCL2 ^*Proteic plasmid transforms the TAP302 cell, and this cell is a BL21pLys S cell, thus its engineered formation that makes the interior oxidation-reduction potential of cell more help disulfide bond that knocks out through thioredoxin reductase.Use this bacterial strain, disulfide bond is formed in the cell, does not need folding step again.

Protocols in Molecular Biology (PCR sudden change and amplification, dna sequencing, restrictive diges-tion) with standard obtains and handles all constructions.In E.coli, produce albumen with one among the clone who comprises correct MCP-1 (P8A) sequence subsequently.Plasmid also is used to transform BL21Star ^TM(DE3) (n ° of C6010-03 of Invitrogen Cat) or BL21DE3 (n ° of 69387-3 of Novagen Cat).

CCL-2, CCL-2 ^*And CCL-2 ^*The method of describing in-P8A such as the former paper is expressed and purification, by the ultrasonotomography step, and cleavage step and chromatographic step (SP-agarose column; Be dissolved in 10mM K with NaCl ₂PO ₄Carry out gradient elution, pH7.5).Merge the peak fraction, be further purified with reversed-phase HPLC (C18 post) with 5 μ m particle diameters and 300  apertures.The acetonitrile eluted protein that comprises 0.1% trifluoroacetic acid that uses gradient to improve; Usually, albumen is at 34 ± 5% acetonitrile place eluting.With they lyophilizing, being dissolved to concentration in 35mM Tris (pH8) is 1mg/ml then, and the amino peptidase of every 1mg albumen and 15 μ g (NJ) at room temperature reacted 36 hours, uses reversed-phase HPLC repurity for Peprotech, Rock Hill by reaction.Amino peptidase is handled and has only been removed the terminal methionine of N-, generates terminal glutamine of N-or the terminal pyroglutamic acid of N-(this changes for not influence of biological activity), and this N-end sequencing by recombiant protein is observed.With the albumen lyophilizing, be dissolved in the water again then, and be stored in the bottle in 80 ℃ with 1-5mg/ml.

A large amount of recombiant proteins, particularly CCL2-P8A also can select another kind of scheme to prepare, and described scheme is that the purification of the cell precipitation that cultivate to obtain for the large scale fermentation that originates in the E.coli bacterial strain designs.The fermentation tank of a common 5L produces the cell precipitation of about weight in wet base 200g, and the fermentation tank of a 50L produces the cell precipitation of about weight in wet base 1.8kg.Purification step described herein is handled the cell precipitation of weight in wet base 200g.

The cell precipitation that thaws, every gram weight in wet base add the 3ml lysis buffer, and (50mM Tris/HCl buffer, (Cat.20092391, Biosolve), it comprises 10mM MgCl to pH8.0 ₂(Cat.63065, Fluka), 5mM benzenecarboximidamide/HCl (Cat.12073, Fluka), 1mM 1, and 4-DL dithiothreitol, DTT (DTT) (Cat.43819, Fluka), 1mM Benzylsulfonyl chloride (PMSF) (Cat.78830, Fluka) * 20mg/LDNase (Fluka) (Cat.DN-25, Sigma)).Suspension homogenizes to obtain the good homogenate that does not contain fragment or grumeleuse with Polytron.All operating under 4 ℃ carried out.The bacterial suspension that homogenizes is transferred to French Press cell machinery fracturer (differential pressure).Operation (Passages) number of times generally is 1500 crust 2-4 time down.Cell rupture is detected by the painted SDS-PAGE of the bright basket of coomassie.

Lysate is assigned in the GSA centrifuge tube, and 10, (16,300 * g) is centrifugal, carried out 90 minutes at 4 ℃ with Sorval RC5C down for 000rpm.After centrifugal, after SDS-PAGE analytically can not detect relevant soluble protein in the clear liquid, abandoning supernatant.With curet from centrifuge tube, shift out the precipitation and transfer in the beaker of weighing in advance, precipitation is weighed.The precipitate with deionized water washing in beaker, adds 5ml water, and stirred 30 minutes at 4 ℃ with magnetic stirring apparatus in every gram precipitation.Suspension is placed the GSA centrifuge tube, and 10, (16,300 * g) is centrifugal, carried out 60 minutes at 4 ℃ with Sorval RC5C centrifuge down for 000rpm.Washing step repeats 3 times.After each centrifugal, after in SDS-PAGE checking supernatant, can not detecting relevant soluble protein, abandoning supernatant.

To precipitate extraction buffer (the 100mM Tris/HCl buffer that (in the inclusion body) is dissolved in prepared fresh, pH8.0 (Cat.20092391, Biosolve), comprise 1mM 1,4-DL dithiothreitol, DTT (DTT) (Cat.43819, Fluka) and 6M guanidine (Guanidium)/HCl (Cat.50950, Fluka), ratio is used for the 25g cell for the 100ml buffer, uses Polytron) in.Solution was heated 30 minutes at 60 ℃, and stir, then cool to room temperature to guarantee singulation.Homogenate is assigned in the Ti45 centrifuge tube, and (100,000 * g) 35, super centrifugal under the 000rpm to use BeckmanL-60.(SpiralCap (Cat.12069, PALL)) filters supernatant, analyzes with SDS-PAGE, and uses Coomassie analysis of protein reagent (Pierce) quantitative according to the guidance of the test kit that is provided with the 0.8-0.2mm filter.

Relevant recombiant protein is hunted down on the FineLine 35 Pilot posts that comprise Source 30RPC resin, and described resin loads according to the explanation that producer (Amersham Pharmacia) provides.After use, the cleaning that provides according to producer is with column regeneration.For the cell precipitation of 100 grams, the high Source 30RPC post of filling 3.5cm diameter * 23em, 220ml (being equivalent to 1 column volume) altogether.Post is installed on the AKTAFPLC (Amersham Pharmacia).Flow velocity is 10ml/min, and maximum pressure is 1Mpa.Before last sample, described post is with the deionized water wash of 2 times of column volumes (440ml).After washing, and the level pad of described post usefulness 5CVs (column volume) (100mM Tris/HCl buffer (Cat.20092391, Biosolve), the HCl of being fuming with 37% (Cat.84426 Fluka) is adjusted to pH7.5) balance.

The lbs (inclusion body) that is dissolved in guanidine/HCl with sample on the flow velocity of 5ml/min to post.With the level pad washing of post, then use buffer A (0.1%TFA trifluoroacetic acid (Cat.28904 is Pierce) with the 99.9% distilled water) washing of 5CV then with 5 times of column volumes (CV).Use to surpass the linear gradient from 0% to 100% of 10CV buffer B (the 0.1%TFA trifluoroacetic acid (and Cat.28904, Pierce), 9.9% distilled water, 90% acetonitrile (UN1648, Baker)) eluting, flow velocity 10ml/min, and 1MPA pressure limit.Collect the 10ml fraction.All detected peaks are analyzed with SDS-PAGE, HPLC, and quantitative with the UV-optical spectroscopy.Merge the fraction that contains desirable proteins, quantitative with the UV-optical spectroscopy.

Protein 10 doubly is diluted to renaturation buffer (100mM Tris-HCl buffer, pH8.0 (Cat.20092391, Biosolve), comprise 0.1mM reduced glutathion (Cat.G-4251, Sigma) and the 0.01mM oxidized form of glutathione (Cat.120 000 250, Acros Organic)) carry out renaturation in, the ultimate density of acquisition is about 0.1mg/ml.The merging thing that in renaturation buffer, dropwise adds Source 30PRC.Very big as fruit volume, this can be undertaken by using peristaltic pump.Solution is spent the night 4 ℃ of stirrings.Owing to do not have the proteic precipitation of renaturation, cause solution often to present muddiness.Ultimate density produces normal recombinant protein (40-50%) in the scope of 0.1-0.4mg/ml in buffer.In initiation material, pH and acetonitrile can not influence the renaturation step.Carry out HPLC after the renaturation, then carry out again folding.

Use High Flow peristaltic pump, use two membrane filtration renaturation solution of forming by pre-filter membrane of 0.8mm and 0.22mm filter membrane.Then, after quantitative, clear solutions is concentrated on Hiload SP Sepharose HP by cation exchange with UV-spectrum.The size of ion exchange column depends on proteic amount.For＜500 milligrams albumen, use 16/10 post (1CV=20ml); For the albumen of 500-1000 milligram, use 26/10 post (1CV=50ml), the albumen for the 1-2 gram uses 50/5 post (1CV=100ml).

Described post loads according to the guidance of manufacturer (Amersham Pharmacia).With the deionized water wash post of 2CV, use cation exchange buffer A (50mM acetic acid (Fluka) is adjusted to pH4.5 with NaOH (the Cat.71690Fluka)) balance of 4CV then.With acetic acid solution is adjusted to pH4.5, conductivity is adjusted to＜10mS.After sample arrives selected post on the flow velocity that protein solution is advised with manufacturer, with the buffer A column scrubber of 5CV.(buffer A comprises 2M NaCl (Cat.71380, Fluka)) eluting to albumen with the buffer B of the linear gradient from 0% to 100% that surpasses 20CV.How many fraction depends on the size of post.All detected peaks are analyzed by SDS-PAGE, HPLC, and quantitative with the UV-optical spectroscopy.After analysis, merge the fraction that contains AS900652, use UV-spectrum quantitative, and analyze with HPLC.

Use methionine amino peptidase (MAP), remove the terminal methionine of N-, then carry out purification step by enzymatic.In brief, at first use cutoff value sample to be dialysed to lysis buffer (35mM Tris/HCl buffer, pH7.5 (Cat.20092391, Biosolve)) as the film pipe of 3.5kD.In 24 hours, change dialysis buffer liquid 3 times.(w: w, enzyme: ratio albumen) joined in the protein solution methionine amino peptidase (MAP) with 1: 10000.Digestion was at room temperature carried out 48 hours.Then by the albumen of foregoing cation exchange purification through digestion.With SDS-PAGE assessment, purity of protein＞98%.

Use AKTA purification system (Pharmacia) to be further purified required albumen.Clean described system 1 hour with 1M NaOH, with aquesterilisa washing, through the buffer of 0.22mm membrane filtration (balance among the 0.1%TFA (trifluoroacetic acid) (Cat.28904, Pierce), 99.9% distilled water).Protein G-25 fine SepharoseXK50/30 post desalination.With the 1M NaOH column scrubber of 1CV, then, use 5CV 0.1%TFA balance then with the aquesterilisa washing of 4CV.Best desalination condition is the desalination on 450ml G-25 fineSepharose of 50-100ml sample.For sample, repeat the desalination step greater than the 100ml volume.Before last sample, sample 0.22mm membrane filtration.Post carries out eluting with the TFA of 1.5CV 0.1% with flow velocity 10ml/min, maximum pressure 1Mpa.The fraction of 10ml is collected in the sterile tube.After the analysis, merge and comprise proteic fraction, under sterilizing state, carry out quantitatively, and analyze with HPLC, SDS-PAGE and mass spectrography with UV-spectrum.

Remaining impurity is gone up at DeltaPrep HPLC (WATERS) with preparation property reversed phase chromatography method (RPC) and is removed.Sample 0.1%TFA acidify, last sample to through buffer A (the equilibrated Vydac C8RPC of 0.1%TFA trifluoroacetic acid (Cat.28904, Pierce) and 99.9% distilled water) (and Cat.208TB101522, Vydac).Use to surpass the linear gradient of 10CV from the buffer B of 0%-100% (the 0.1%TFA trifluoroacetic acid (and Cat.28904, Pierce), 99% acetonitrile (UN1648, Baker)) eluted protein, flow velocity is 25ml/min, pressure limit is 700 crust.All detected peaks are analyzed by SDS-PAGE, HPLC, and quantitative with the UV-optical spectroscopy.After analysis, merge the fraction that contains AS900652, quantitative with UV-spectrum, aliquot and lyophilizing as required.Albumen is kept at-20 ℃ or-80 ℃.

Recombiant protein is quantitative by the UV-optical spectroscopy, and use UV-VIS spectrophotometer (the Uvikon system, KONTRON).(QS 1.000, HELLMA) as the buffer contrast, in other cuvette sample are housed with a quartz cuvette.According to using Protparam, the extinction coefficient that Expasy obtains from the aminoacid composition detect in the scanning of 350nm to 240nm scope, use the light absorption of 280nm to come quantification.

(Cat.NP0301 Invitrogen) carries out SDS-PAGE and analyzes to use NuPAGE 10% gel.(cat.LC2676, Invitrogen) middle dilution was 2 times, 95 ℃ of heating 5 minutes at sample buffer for sample.Use the standard protein band as molecular weight standard.Sample is in suitable hole on the molecular weight standard solution of 10 μ l and the protein sample of 20 μ l, electrophoretic buffer be MES (Cat.NP0002, Invitrogen).At 200V, (PowerEase 500, Invitrogen) in 35 minutes for electrophoretic transfer under 12mA and the 25W condition according to the guidance of manufacturer.

The NuPAGE gel is containing 10% acetic acid, uses 0.1%R250 coomassie brilliant blue staining 30 minutes in the distilled water of 30% methanol, slowly shaking down, at 10% acetic acid, goes in the distilled water of 30% methanol-dye again, and is colourless until background level.Then gel is washed in water for several times, be embedded in the drying solution (drying solution) that is clipped in the middle by two-layer cellophane (Invitrogen) (Invitrogen) in 10 minutes, apply small pressure then and make the gel formation thin layer.Can be randomly, (Cat.LC6065's gel Invitrogen) dyes with SimplyBlue Safe dyeing scheme.

(0.2cm diameter * 22cm) be used is used the 0.1%TFA balance with analyzing C8 Aquapore RP-3007m in the Alliance HPLC system that is provided by WATERS.Inject the acidifying in advance 0.1% final concentration TFA of 10-50mg.With the 25-50% acetonitrile gradient eluting that surpasses 20CV.

The feature of recombiant protein obtains the correct albumen of N-end sequence QPDAINAAVT by mass spectral analysis and the checking of N-terminal sequence analysis from the material of purification.

The albumen of the main type that obtains is 8655Da, conforms to the proteic Theoretical Mass with two disulfide bond.Observed second type albumen, than the low 17Da of aforementioned albumen, to become pyroglutamic acid corresponding with the terminal glutamine residue of N-.The existence of this modification is for the not influence of proteic activity.

Embodiment 2 test cell lines

Material and method

The inductive peritoneal cell of chemotactic factor is raised test

The female Balb/C mice in 8-12 week (Janvier France) is raising under normal growth of animal condition, described condition be 12 hours of standard the daytime/cycle at night and freely obtain food and water.Comprise the saline (NaCl 0.9% (w/v) of the no LPS of sterilization) of the group peritoneal injection 200 μ l of 3-6 mice, or comprise the described solution of CCL2 or CCL2-P8A (per injection 10 μ g).In order to study the inhibition effect that CCL2-P8A raises for the inductive peritoneal cell of CCL-2, at peritoneal injection CCL2 preceding 30 minutes, first intraperitoneal gave these molecules.All molecules give with foregoing concentration with in aforesaid buffer (saline).Behind last injection CCL-2 or CCL-2-P8A 4 hours, put to death mice.

The inductive peritoneal cell of thioglycolate salt (thioglycollate) is raised open (Mishell B, 1980).In brief, preparation TGA base status (medium), method are that the dehydration TGA base status (Becton Dickinson) with 30g is suspended in 1 liter the cold distilled water, are heated to boiling then to dissolve powder fully.Then the substrate five equilibrium is assigned to the 100ml bottle, and autoclaving.After cooling, with substrate in the dark, store at least one month under the room temperature.At the 1st day, at CCL2 ^*-P8A gives back 30 minutes, and the 3% TGA saline solution of peritoneal injection 200 μ l is raised with inducing cell in 3 groups of mices.(the 2nd day, the 3rd day, the 4th day) gave CCL2 every day in 3 days afterwards ^*Use dexamethasone (Sigma) as positive control, it is with the 10mg/kg peritoneal injection.Mice was put to death at the 5th day.

Cell in the test that is evaluated at the front with peritoneal lavage as described below is raised.By CO in the rising lucite box ₂Concentration, make mice death by suffocation.Clean mouse skin with 70% ethanol.Remove the skin of skin, expose peritoneum.Peritoneal cavity is incorporated in liquid the 15ml polystyrene Falcon pipe (Becton Dickinson) that places on ice with 5ml ice-cold PBS (phosphate buffered saline (PBS)) lavation 3 times.The subsidiary peritoneal cavity of gently massaging during each lavation.Irrigating solution is centrifugal at 425xg, removes supernatant, and remaining cell precipitation is resuspended among the 1ml PBS by leniently repeatedly inhaling to put, and 10 μ l cell suspending liquids dye with 90 μ l trypan blues, and total cell number is counted every 1mm with the Neubauer hemocytometer ²Count 4 zones.According to the instruction that hemocytometer carries, calculate the meansigma methods of 4 countings, multiply by dilution gfactor 10, multiply by the cell number of the every μ l of 10 acquisitions again.Last total value multiply by 1000 (to equaling 1ml), with the total cell number that obtains to reclaim.

The result

The reorganization one-tenth acquaintance CCL2/MCP-1 and be known as the mutant of CCL2* and be known as CCL2-P8A and CCL2 accordingly ^*The obligate monomer mutant of-P8A (Fig. 1) is expressed among the E.coli.

Document clearly illustrates, the formation of CCL2 dimer has been blocked in P8A sudden change among the CCL2, be not attached on the cell of expressing its receptor or be attached on the Virus receptors albuminoid and do not influence it, simultaneously at correlation test (referring to Paaavola CD etc., (1+9-76) MCP-1 in 1998 the table 1); Alexander JM etc., 2002) in, do not show the activity of known CCL2 antagonist yet.

In test cell line, the obligate monomeric form of CCL2 presents special and unexpected characteristic.Raise in the test at peritoneal cell, compare CCL2 with natural CCL2 ^*-P8A and CCL2-P8A can not raise cell (Fig. 2).And these molecules can suppress CCL2 inductive (Fig. 3 A) and thioglycolate salt inductive (Fig. 3 B) macrophage is raised in dose-dependent mode.In one test of back, CCL2-P8A presents and the same effectiveness of positive control (dexamethasone, a kind of known antiinflammation compound).

Embodiment 3: the characteristic of CCL2-P8A in animal disease model

Material and method

The inductive pneumonia model of ovalbumin

The inductive pneumonia model of ovalbumin (Blyth D1 etc., 1996) method is as disclosed made.The mice group of 6 mices is by peritoneal injection 10 μ g egg white powder sensitizations, described egg white powder is deposited in the solution of 2mg aluminium hydroxide 2% (Serva), cumulative volume 200 μ l, it is by mixing 25 μ l ovalbumins (2mg/ml) among the 0.9%NaCl (saline) that does not have LPS at 725 μ l, and 250 μ l aluminium hydroxide also made 4 ℃ of precipitations in 3-4 hour.After sensitization 15 days,,, 6 mices are handled and attack by the 15 μ g ovalbumins that intranasal is dissolved in the 50 μ l saline sucking under the anesthesia (isoflurane) from the 15th day to the 19th day.The each attack gave CCL2-P8A (each peritoneal injection 200 μ l, 10 μ g) in preceding 30 minutes.Peritoneal lavage assessment cell is raised, and cell counting is as described in the embodiment 2.

EAE (experiment autoimmune encephalomyelitis) model

(selected strain has been reported the sensitivity for EAE available from Charles River Italy; SahrbacherUC etc., 1998) the C57BI/6 mice wherein contains 200 μ g MOG35-55 peptide (Neosystem by comprising, Strasbourg, France) and comprise the complete Freund ' s adjuvant (CFA of the mycobacterium tuberculosis (mycobacterium tuberculosis) of 0.5mg, Difco, Detroit, Emulsion U.S.A.) subcutaneous (s.c.) are injected in the left side abdomen by immunity (the 0th day).Intraperitoneal (i.p.) injection immediately afterwards be dissolved in 400 μ l buffer (0.5M NaCl, 0.017%Triton X-100,0.015M Tris, 500ng pertussis toxin, PT pH7.5) (List BiologicalLab, Campbell, CA, U.S.A.).At the 2nd day, give the 2nd i.p. injection of animal 500ng pertussis toxin, PT.At the 7th day, give the MOG in CFA of mice 200 μ g the 2nd time _35-55Peptide, s.c. are injected in the right side abdomen.From about 8-10 days, described step caused enhanced gradually paralysis, from tail, extends to forelimb.Since the 7th day, detect these animals individually by following clinical score method and whether have paralysis:

0=does not have the disease sign

0.5=part tail paralysis

1=tail paralysis

1.5=the monolateral back acroparalysis of tail paralysis+part

Weak or the part back acroparalysis of 2=tail paralysis+hind leg

2.5=tail paralysis+part back acroparalysis (lowered pelvi)

3=tail paralysis+complete back acroparalysis

3.5=tail paralysis+back acroparalysis+incontinence fully

4=tail paralysis+back acroparalysis+forelimb weakness or partial paralysis

5=is dying or dead

After immunity the 7th day,, and handle 21 days (every processed group 10-12 animal) continuously with chemical compound or every animal of vehicle treated.Interferon beta and CCL2-P8A be respectively by s.c. or i.p. injection, once a day, is dissolved in the PBS of 10ml/kg with the dosage shown in scheming.

The super quick model of tardy property contact

It is super quick to measure contact as previously described (Garrigue JL etc., 1994) with the mouse ear expansion test.In brief, by being applied to 0.5%2 of 25 μ l in acetone/olive oil (4: 1), 4-dinitrofluorobenzene (DNFB; Sigma Chemical Co.) solution makes the local pre-sensitizing of mice to plucked abdominal part.After 5 days, the 0.2%DNFB that will be in 20 μ l in the identical carrier is applied on the auris dextra, and independent carrier is applied on the left ear.Mice (every group of n=6) is handled once every day, applies 0.05,0.5 or the CCL2-P8A of 5mg/kg (being respectively 1,10 or 100 μ g/ mices) at the 5th day intraperitoneal, dexamethasone (1mg/kg), or in matched group, only apply PBS.Described processing was carried out before DNFB attacks in 30 minutes.Measure ear thickness with dial thickness gauge (Mitutoyo Corp.), by deducting the value before attacking with the value after attacking, and by further deducting the expansible value of the offside ear that any observed carrier attacks, the expansion of estimation ear.

The result

CCL2-P8A tests in the animal model of inflammation and autoimmune disease as the potential therapeutic activity of chemokine antagonists.

CCL2 ^*-P8A tests in the disease model of the inductive pneumonia of ovalbumin.In the model of the allergy pneumonia of this classics, in the sensitization stage, mice is used the ovalbumin sensitization, with aluminium hydroxide as adjuvant to increase immunoreation, give ovalbumin by intranasal then and attacked continuous 5 days, wherein intraperitoneal gives CCL2-P8A in all stage.Also in this case, CCL2-P8A can suppress cell and raises (Fig. 4).

In second kind of model, CCL2-P8A adopts EAE (experiment autoimmune encephalomyelitis) model to test, the EAE model is a kind of known multiple sclerosis model, be used to verify that the antagonist of chemotactic factor (comprising CCL2) is used for the treatment of autoimmune, people central nervous system's inflammatory demyelinating disease (Mahad DJ and Ransohoff RM, 2003, lzikson L etc., 2002).CCL2-P8A tests in presenting the described disease of medium or the order of severity, with after inducing the compound treatment of EAE, then assesses by clinical score.Each group of two treated animals is divided into 5 subgroups: use not commensurability CCL2-P8A to handle for 3 groups in them, other 2 groups are used as negative control (only using vehicle treated) or as positive control (using interferon beta, a kind of common treatment product that is used for the treatment of multiple sclerosis).The development of animal state compares based on the clinical score that records in the processing cycle (21 days).In two kinds of disease models, the state that gives to have improved significantly statistically animal of CCL2-P8A (dosage is reduced to 0.15mg/kg).Use the decline of the observed clinical score of CCL2-P8A to treat viewed scoring quite (Fig. 5) with using interferon beta at least.

Another kind of disease model, contact hypersensitivity model is used to assess CCL2-P8A for the potential curative effect by the cell-mediated scytitis of T.Contact hypersensitivity (CHS) is a kind of Langerhans' cells (LC) skin immunization reaction that rely on, that T is cell-mediated, it reflects in the body (the antigenic picked-up of epidermis (epicutaneous) that peaks of LC activity, transfer to lymph node, and submission antigen is to natural T cell).This model can perform well in evaluating chemical compound, is used for dermatological indication such as psoriasis and allergic contact dermatitis (Xu H etc., 1996).It comprises a sensitization stage and a follow-up antigen attack process, and it causes scytitis, forms edema and Premeabilisation of cells in skin.Edema can be by measuring with caliper in attack position (mouse ear).Proved that now chemotactic factor (particularly CCL) participates in these over-reactive advancing of disease (Mitsui G etc., 2003; Mizumoto N etc., 2001).Giving CCL2-P8A here, cause expansion ratio to be handled and use the viewed expansion of known antiinflammation compound (dexamethasone) to reduce to some extent one day after with preceding 30 minutes intraperitoneal of antigen (being DNFB) attack.Control mice is by attack obtaining with antigen, but through or without before sensitization, so inflammation and edema that the T cell relies on form or do not form (Fig. 6).

Therefore, (wherein said variant is formed by at least one aminoacid replacement the monomer variant of the CC-chemotactic factor of formation homodimer, this replacement has changed the form of the hydrogen bond on the dimerization interface) in vivo cell raise in the test and be the inhibitor that chemokine mediated cell is raised at the animal model that is used for human diseases, mean that this is a kind of new strategy that is used to generate chemokine variants, it can be used for pharmaceutical compositions and is used for the treatment of in the method.

The available form of embodiment 4:CCL2-P8A

The alternate forms of the chemokine variants that discloses previously can known in the artly generate as the sudden change that improves characteristic by introducing.

One or more simple amino acid whose replacements and/or adding can be introduced in the diverse location (Fig. 7 A) of CCL2-P8A.CCL2-P8A can be expressed as the mature peptide that loses natural glutamine (glutammine) N-terminal residue, or by before the glutamine of N-end, add little residue (as alanine or glycine) make as described in residue can not keep exposed state, and can spontaneously not be converted to pyroglutamic acid form (Gong J and Clark-Lewis I, 1995).CCL-P8A also can be mutated into by realizing specific pegylation reaction with the 5th cysteine.These Pegylation sites can be to be incorporated into internal amino acid (such as in asparagine 14 or 17, even on 8, be convenient to single modification and can allow singulation and Pegylation simultaneously) or in (by behind natural C-terminal threonine, directly adding cysteine) on the C-end.

The further variant of CCL2-P8A can obtain to the constant region of immunoglobulin (stability of known raising recombiant protein in circulation and the protein structure domain of effect) by merging described sequence.The gained fusion rotein can directly be expressed by mammalian cell (as CHO or HEK293 cell) with suitable expression vector, makes that fusion rotein is secreted in culture medium.In a preferred mode, the nucleotide sequence of encoding mature CCL2-P8A can be cloned in the expression vector, its 5 ' end is fused on the nucleotide sequence of coding people CCL2 signal sequence, and its 3 ' end is fused to coding human normal immunoglobulin λ heavy chain IgG1 (NCBI accession number: CAA75302) on the nucleotide sequence of constant region (fragment 246-467).The carrier that obtains can be used to transform CHO or HEK293 cell line, and can select stably to express and secrete the N-end and have the clone (Fig. 7 B) that CCL2-P8A, C-end have the recombination fusion protein of IgG1 sequence.This clone can be used to enlarge to produce, and from culture medium the purification of Recombinant fusion rotein.Perhaps, the position of the nucleic acid of coding human normal immunoglobulin's λ heavy chain IgG1 constant region (fragment 243-474) and CCL2-P8A can exchange, and the albumen of acquisition is end user CCL2 signal sequence still, or any other signal sequence is expressed and secreted.

Table 1

Aminoacid	Synonym aminoacid	Preferred synonym aminoacid
			Ser	Gly，Ala，Ser，Thr	Thr，Ser
Arg	Asn，Lys，Gln，Arg，His	Arg，Lys，His
			Leu	Phe，Ile，Val，Leu，Met	Ile，Val，Leu，Met
Pro	Pro，Ala，Ser，Thr	Pro
			Thr	Gly，Ala，Ser，Thr	Thr，Ser
Ala	Gly，Thr，Ser	Gly，Ala
			Val	Met，Phe，Ile，Leu，Val	Met，Ile，Val，Leu
Gly	Ala，Thr，Ser，Gly	Gly，Ala
			Ile	Phe，Ile，Val，Leu，Met	Ile，Val，Leu，Met
Phe	Trp，Phe，Tyr	Tyr，Phe
			Tyr	Trp，Phe，Tyr	Phe，Tyr
Cys	Ser，Thr，Cys	Cys
			His	Asn，Lys，Gln，Arg，His	Arg，Lys，His
Gln	Glu，Asn，Asp，Gln	Asn，Gln
			Asn	Glu，Asn，Asp，Gln	Asn，Gln
Lys	Asn，Lys，Gln，Arg，His	Arg，Lys，His
			Asp	Glu，Asn，Asp，Gln	Asp，Glu
Glu	Glu，Asn，Asp，Gln	Asp，Glu
			Met	Phe，Ile，Val，Leu，Met	Ile，Val，Leu，Met
Trp	Trp，Phe，Tyr	Trp

Table 2

Aminoacid	Synonym aminoacid
		Ser	D-Ser, Thr, D-Thr, not-and Thr, Met, D-Met, Met (O), D-Met (O), L-Cys, D-Cys
Arg	D-Arg, Lys, D-Lys, height-Arg, D-height-Arg, Met, Ile, D-.Met, D-Ile, Orn, D-Orn
		Leu	D-Leu，Val，D-Val，AdaA，AdaG，Leu，D-Leu，Met，D-Met
Pro	D-Pro, L-1-Thiazolidine (thioazolidine)-4-carboxylic acid, D-or L-1- azoles alkane-4-carboxylic acid
		Thr	D-Thr, Ser, D-Ser, not-and Thr, Met, D-Met, Met (O), D-Met (O), Val, D-Val
Ala	D-Ala，Gly，Aib，B-Ala，Acp，L-Cys，D-Cys
		Val	D-Val，Leu，D-Leu，Ile，D-Ile，Met，D-Met，AdaA，AdaG
Gly	Ala，D-Ala，Pro，D-Pro，Aib，.β-Ala，Acp
		Ile	D-Ile，Val，D-Val，AdaA，AdaG，Leu，D-Leu，Met，D-Met
Phe	D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, trans-3,4, or 5-phenyl proline, AdaA, AdaG, cis-3,4, or 5-phenyl proline, Bpa, D-Bpa
		Tyr	D-Tyr，Phe，D-Phe，L-Dopa，His，D-His
Cys	D-Cys，S--Me--Cys，Met，D-Met，Thr，D-Thr
		Gln	D-Gln，Asn，D-Asn，Glu，D-Glu，Asp，D-Asp
Asn	D-Asn，Asp，D-Asp，Glu，D-Glu，Gln，D-Gln
		Lys	D-Lys, Arg, D-Arg, height-Arg, D-height-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn
Asp	D-Asp，D-Asn，Asn，Glu，D-Glu，Gln，D-Gln
		Glu	D-Glu，D-Asp，Asp，Asn，D-Asn，Gln，D-Gln
Met	D-Met，S--Me--Cys，Ile，D-Ile，Leu，D-Leu，Val，D-Val

List of references

Alexander JM etc., Cell, 111; 343-356,2002.

Baggiolini M etc., Annu Rev Immunol, 15:675-705,1997.

Baggiolini M，J Intern Med，250：91-104，2001.

BlythDI etc., Am J Respir Cell Mol Biol., 14:425-438,1996.

Brown A etc., J Pept Sci 2:40-46,1996.

Cleland JL etc., Curr Opin Biotechnol, 12:212-219,2001.

ColemanRA etc., Drug Disc Toay, 6:1116-1126,2001.

Dougherty DA，Curr Opin Chem Bio，4：645-652，2000.

Fernandez EJ and Lolis E, Annu Rev PharmacolToxicol, 42:469-499,2002.

Garrigue JL etc., Contact Dermatitis, 30:231-237,1994.

Godessart N and Kunkel SL, Curr Opin Immunol, 13:670-675,2001.

Golebiowski A etc., Curr Opin Drug Discov Devel, 4:428-434,2001.

Gong J and Clark-LewisI, J Exp Med 181:631-640,1995.

Gosling J etc., J Clin Invest, 103:773-778,1999.

Greenwald RB etc., Adv Drug Deliv Rev, 55:217-50,2003.

Gu L etc., Chem Immunol, 72:7-29,1999.

Gu L etc., Nature, 404:407-411,2000.

Harris JM and Chess RB, Nat Rev Drug Discov, 2:214-21,2003.

Handel T etc., Biochemistry, 35:6569-6584,1996.

Hemmerich S etc., Biochemistry, 38:13013-13025,1999.

Hruby VJ and Balse PM, Curr Med Chem, 7:945-970,2000.

Izikson L etc., Clin Immunol, 103:125-31 2002.

" FEBS Lett, 395:277-282,1996. such as Kim KS

Li AP，Drug Disc Today，6：357-366，2001

Loetscher P and Clark-Lewis I, J Leukoc Biol, 69:881-884,2001.

Lu B etc., J Exp Med, 187:601-608,1998.

Lubkowski J etc., Nat Struct Biol, 4:64-69,1997.

Luo B and Prestwich GD, Exp Opin Ther Patents, 11:1395-1410,2001.

Mishell B，Immunopharm，2：233-245.1980.

Mahad DJ and Ransohoff RM, Semin Immunol, 15:23-32,2003.

Mitsui G etc., Immunol Lett, 86:191-7,2003.

Mizumoto N etc., Immunobiology, 204:477-93,2001.

Murphy LR etc., Protein Eng, 13:149-152,2000.

Nilsson J etc., Protein Expr Purif, 11:1-16,1997.

Paavola C etc., J Biol Chem, 273:33157-33165,1998.

Pillai 0 and Panchagnula R, Curr Opin Chem Biol, 5:447-451,2001.

Proudfoot A etc., ProcNatl Acad Sci U S A, 100:1885-1890,2003.

Rogov Sl1 and Nekrasov AN, Protein Eng, 14:459-463,2001.

Sahrbacher UC etc., Eur J Immunol, 28:1332-8,1998.

SchwarzMK and Wells TN, Curr Opin Chem Biol, 3:407-417,1999.

Seet BT etc., ProcNatl Acad Sci U S A, 98:9008-9013,2001.

Steitz SA etc., FEBS Lett, 430:158-164,1998.

Villain M etc., Chem Biol, 8:673-679,2001.

Yoshimura T etc., FEBS Lett, 244:487-493,1989.

Xu H etal，J Exp Med，183：1001-12，1996.

Zhang Y etc., Methods, 10:93-103,1996.

Sequence table

＜110〉Applied Research Systems ARS Holding N.V (Applied Research Systems ARS holding)

＜120〉therapeutic of chemokine variants is used

<130>WO933

<160>5

<170>PatentIn version 3.0

<210>1

<211>76

<212>PRT

＜213〉homo sapiens

<220>

＜223〉people CCL2

<400>1

Gln Pro Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr

65 70 75

<210>2

<211>76

<212>PRT

＜213〉synthetic construction

<220>

＜223〉people CCL2-P8A

<400>2

Gln Pro Asp Ala Ile Asn Ala Ala Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Thr 6ln Thr Pro Lys Thr

65 70 75

<210>3

<211>76

<212>PRT

＜213〉synthetic construction

<220>

＜223〉people CCL2*

<400>3

Gln Pro Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys 6lu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Ile

50 55 60

Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr

65 70 75

<210>4

<211>76

<212>PRT

＜213〉synthetic construction

<220>

＜223〉people CCL2*-P8A

<400>4

Gln Pro Asp Ala Ile Asn Ala Ala Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Ile

50 55 60

Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr

65 70 75

<210>5

<211>331

<212>PRT

＜213〉synthetic construction

<220>

＜223〉people CCL2-P8A_IgG1 fusion rotein

<400>5

Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu Ile Ala Ala Tht

1 5 10 15

Phe Ile Pro Gln Gly Leu Ala Gln Pro Asp Ala Ile Asn Ala Ala Val

20 25 30

Thr Cys Cys Tyr Asn Phe Thr Asn Arg Lys Ile Ser Val Gln Arg Leu

35 40 45

Ala Ser Tyr Arg Arg Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala Val

50 55 60

Ile Phe Lys Thr Ile Val Ala Lys Glu Ile Cys Ala Asp Pro Lys Gln

65 70 75 80

Lys Trp Val Gln Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln Thr

85 90 95

Pro Lys Thr Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

100 105 110

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

115 120 125

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

130 135 140

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

145 150 155 160

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

165 170 175

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

180 185 190

Leu His Asn Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

195 200 205

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

210 215 220

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

225 230 235 240

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

245 250 255

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Gln Gly Gln Pro Glu

260 265 270

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

275 280 285

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

290 295 300

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

305 310 315 320

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

Claims (23)

1. comprise of the application of the polypeptide of SEQ ID NO:2 as medicine.

2. application as claimed in claim 1 is characterized in that, described polypeptide also contains isoleucine at the 64th of SEQ ID NO:2.

3. application as claimed in claim 1 or 2, its feature also be, described polypeptide is not containing sudden change with SEQ ID NO:2 and the corresponding sequence of SEQ ID NO:4 the 9th, 10 or 13.

4. application as claimed in claim 1 or 2, its feature also be, described polypeptide is with SEQ ID NO:2 and the corresponding sequence of SEQ ID NO:4:

A) the 8th, 14,17 or 77 contains cysteine; Or

B) the 1st contains alanine or glycine.

5. as each described application of claim 1-4, its feature is that also described polypeptide contains human immunoglobulin heavy chain's constant region.

6. the polypeptide that contains SEQ ID NO:2 is used for the treatment of application in the medicine of autoimmune disease, inflammatory diseases or infectious disease in preparation.

7. as the described application of claim 65, it is characterized in that described polypeptide also contains isoleucine at the 64th of SEQ ID NO:2.

8. as claim 6 or 76 described application, its feature also is, described polypeptide is not containing sudden change with SEQ ID NO:2 and the corresponding sequence of SEQ ID NO:4 the 9th, 10 or 13.

9. as claim 6 or 7 described application, its feature also is, described polypeptide is with SEQ ID NO:2 and the corresponding sequence of SEQ ID NO:4:

A) the 8th, 14,17 or 77 contains cysteine; Or

B) the 1st contains alanine or glycine.

10. as each described application of claim 6-9, its feature is that also described polypeptide contains human immunoglobulin heavy chain's constant region.

11. application as claimed in claim 6, it is characterized in that described disease is selected from: arthritis, rheumatic arthritis (RA), psoriatic arthritis, osteoarthritis, systemic lupus erythematosus (sle) (SLE), systemic sclerosis, scleroderma, polymyositis, glomerulonephritis, cystic fibrosis, fibroid degeneration, irritated or super quick disease, dermatitis, asthma, chronic pulmonary is blocked disease (COPD), enteritis (IBD), Crohn ' s disease, ulcerative colitis, multiple sclerosis, cancer, septic shock, virus or HIV infect, transplant, airway inflammation, anti-host disease of transplantability (GVHD) and arteriosclerosis.

12. application as claimed in claim 7, it is characterized in that described disease is selected from: arthritis, rheumatic arthritis (RA), psoriatic arthritis, osteoarthritis, systemic lupus erythematosus (sle) (SLE), systemic sclerosis, scleroderma, polymyositis, glomerulonephritis, cystic fibrosis, fibroid degeneration, irritated or super quick disease, dermatitis, asthma, chronic pulmonary is blocked disease (COPD), enteritis (IBD), Crohn ' s disease, ulcerative colitis, multiple sclerosis, cancer, septic shock, virus or HIV infect, transplant, airway inflammation, anti-host disease of transplantability (GVHD) and arteriosclerosis.

13. application as claimed in claim 11 is characterized in that, described disease is a multiple sclerosis.

14. application as claimed in claim 12 is characterized in that, described disease is a multiple sclerosis.

15.SEQ ID NO:2 and human immunoglobulin heavy chain's constant region merge the fused polypeptide aminoacid sequence shown in the SEQ ID NO:5 that forms.

16. the nucleotide sequence of the fused polypeptide shown in the coding SEQ ID NO:5.

17. the fusion rotein method of preparation claim 15 comprises:

A) nucleic acid coding sequence of ripe CCL2-P8A is cloned in the expression vector, its 5 ' terminal nucleotide sequence with coding people CCL2 signal sequence is merged, and 3 ' terminal nucleotide sequence with coding human normal immunoglobulin's λ heavy chain IgG1 constant region (243-474 section) merges;

B) carrier that obtains is transformed into CHO or HEK293 cell line;

C) select stably express and secretion to have the recombination fusion protein that CCL2-P8A and C-end have the IgG1 sequence at its N-end;

D) the described fusion rotein of purification from culture medium.

18. be used to screen the method for obligate monomer antagonist chemokine variants as herein described, comprise:

A) in CCL2, produce the simple point mutation of preventing its dimerization ability;

B) identify the external described variant that is incorporated into receptor and demonstrates antagonism;

C) from the cohort that (b) identifies, further identify the described variant that the inhibition peritoneal cell is raised.

19. a pharmaceutical composition, it contain form homodimer chemotactic factor monomer variant as active component, wherein said variant comes from least one aminoacid replacement of described chemotactic factor dimerization change hydrogen bond pattern at the interface.

20. pharmaceutical composition as claimed in claim 19 is characterized in that, described monomer variant is selected from:

a)CCL2-P8A(SEQ ID NO：2)；

b)CCL2*-P8A(SEQ ID NO：4)；

C) (a) or active mutant (b); Or

D) comprise (a) and (b) or (c) and the polypeptide that belongs to the aminoacid sequence of the protein sequence except that described chemotactic factor.

21., it is characterized in that described monomer variant is active fragment, precursor, salt, derivant, complex or conjugate form as claim 18 or 19 described pharmaceutical compositions.

22. a treatment or the method for preventing autoimmune disease, inflammatory diseases or infectious disease, the monomer variant that comprises the chemotactic factor of the formation homodimer of using effective dose, wherein said variant come from least one aminoacid replacement of described chemotactic factor dimerization change hydrogen bond pattern at the interface.

23. method as claimed in claim 21 is characterized in that, described monomer variant is selected from

a)CCL2-P8A(SEQ ID NO：2)；

b)CCL2*-P8A(SEQ ID NO：4)；

C) (a) or active mutant (b); Or

D) comprise (a) and (b) or (c) and the polypeptide that belongs to the aminoacid sequence of the protein sequence except that described chemotactic factor.

Images